Opioid peptides of the enkephalin family are widely distributed within the central and peripheral nervous systems and are thought to participate in mechanisms relating to pain and analgesia as well as in many other neuronal pathways. The high concentrations of enkephalins found in the adrenal medulla and the presence of circulating enkephalin-immunoreactive peptides are suggestive of a hormonal role for adrenomedullary enkephalins which may relate to the known cardiovascular effects of synthetic opiate compounds. At present the biosynthetic mechanisms responsible for enkephalin production are poorly understood. The proposed project will detail the posttranslational processing steps required for the production of the penta- to octapeptides met5-enkephalin, leu5-enkephalin, met5-enkephalin-arg6-gly7-leu8 and met5-enkephalin-arg6-phe7, using adrenomedullary chromaffin cell cultures as a model system. These studies will be accomplished through continuous and pulse-chase labelling experiments involving immunoprecipitation with antisera known to recognize different molecular weight classes of immunoreactive enkephalins. Potential posttranslational modifications of proenkephalin such as sulfation, phosphorylation, and amidation will be explored. In addition, the project will examine the biosynthesis of proenkephalin-derived peptides during treatment with drugs and hormones which act to increase adrenal enkephalins. The release of the various proenkephalin-derived peptides from chromaffin cell cultures will also be investigated. Information gained through these studies on the biosynthetic pathway and possible regulatory mechanisms associated with the production of adrenal enkephalins should serve not only as a basis for future investigations of enkephalin metabolism within the brain, but also aid in the study of the role of blood borne enkephalins as hormonal regulators.